Inherited blood disorders are especially favorable targets for therapeutic genome editing in that ex vivo modification of patient hematopoietic stem cells (HSCs) followed by autologous transplantation can result in lifelong recovery of normal blood cell production. Recently we developed an improved version of SpCas9 (3xNLS-SpCas9) and an efficient electroporation protocol for genome editing of CD34+ hematopoietic stem and progenitor cells (HSPCs) using SpCas9 ribonucleoprotein (RNP) that leads to highly efficient on-target gene modification, preservation of HSC function and undetectable off-target editing. In principle , homologous recombination (HR) or base editing could be harnessed for the precise correction of disease-associated mutations. However, the requirement for co-delivery of donor template sequence, the cell cycle dependence of HR-based gene repair, and the competing nonhomologous end joining/microhomology mediated end joining mutagenic repair pathways complicate achieving efficient HR in HSCs. Base editing Is currently limited in its targeting range with uncertainty about potential genotoxicity and HSC efficiency. Nuclease-induced predictable end-joining repair (with indels) is a highly efficient means of gene modification, and could itself be therapeutic depending on the allelic outcome. This strategy may be particularly effective for noncoding mutations that impact regulatory elements, such as those that dictate the pattern of mRNA splicing. We hypothesize that genome editing, by directing efficient non-templated end-joining DNA repair in HSCs, could restore gene expression and provide durable therapy for inherited blood disorders associated with splicing mutations. Two of the most common mutations associated with transfusion-dependent ?-thalassemia are HBB IVS1- 11OG>A and IVS2-654C> T which introduce intronic aberrant splice acceptor and donor sites respectively. Using SpCas9 and LbCas12a RNPs, we have successfully disrupted these inappropriate regulatory elements in HSPCs from multiple patient donors. The erythrocytes differentiated in vitro from these nuclease-treated cells display robust increase in normally spliced HBB mRNA and restored adult hemoglobin (HbA) expression, suggesting that this is a potent strategy for therapeutic development. In Aims 1 & 2 we will develop Cas9 and Cas12a editing reagents for these splicing mutations through nuclease optimization, unbiased genome-wide off-target analysis, and assessment of HSC editing rates through xenoengraftment of edited ?-thalassemia patient HSPCs. In Aim 3, we will develop efficient strategies for the non-templated gene editing repair of splice junction disrupting mutations for the IVS2+2T>C mutation in SBOS commonly associated with Shwachman Diamond syndrome. The successful completion of these studies w/1 define editing approaches for the efficient HSC repair of a range of pathogenic splicing mutations that impact hematopoiesis and enable the development of targeted reagents based on existing nuclease platforms for definitive gene therapy.